The monohydroxy bile acid, taurolithocholate (TLC), causes cholestasis in vivo and in isolated perfused livers. It is also cholestatic in vitro and, in this study using isolated rat hepatocyte couplets, causes a reduction of the accumulation of (fluorescent) bile acid in the canalicular vacuoles (cVA) of this polarized cell preparation. The hepatoprotective bile acid, tauroursodeoxycholate (TUDCA), partially protects against the action of TLC when added at the same time. It also partially reverses the cholestatic effect if added after the cells have been exposed to TLC. A second hepatoprotective compound, S-adenosyl-L-methionine (SAMe) also not only partially protects against the action of TLC when added at the same time, but it too is able to partially reverse the cholestatic effect. Neither hepatoprotective agent is fully effective alone, but their effects are additive. In combination, a full restoration of cVA is observed in moderate cholestasis, but not in severe cholestasis. We discuss briefly some possible mechanisms involved in the additive mode of action of both hepatoprotective compounds. In summary, we show for the first time that SAMe and TUDCA can exert an additive effect in the amelioration of TLC-induced cholestasis in isolated rat hepatocyte couplets. This finding may be of possible clinical relevance.